Crystal Structure of a Subtilisin Homologue, Tk-SP, from Thermococcus kodakaraensis: Requirement of a C-terminal β-Jelly Roll Domain for Hyperstability
Crystal Structure of a Subtilisin Homologue, Tk-SP, from Thermococcus kodakaraensis: Requirement of a C-terminal β-Jelly Roll Domain for Hyperstability
复制标题
DOI：
10.1016/j.jmb.2010.05.064
复制
发表时间：
2010-07-23
影响因子：
5.6
通讯作者：
Kanaya, Shigenori
中科院分区：
文献类型：
--
作者：
Foophow, Tita;Tanaka, Shun-ichi;Kanaya, Shigenori
Tk-SP是来自Thermococcus kodakaraensis的超热稳定的枯草杆菌蛋白酶样丝氨酸蛋白酶，并且由其前体（Pro-Tk-SP）与N-和C-前肽自动加工。在2.0埃分辨率下测定缺乏C-前肽的Pro-Tk-SP的活性位点突变体ProN-Tk-S359 A的晶体结构。ProN-Tk-S359 A由N-前肽、枯草杆菌蛋白酶和β-果冻卷结构域组成。两个Ca 2+离子结合到β-果冻卷结构域。不含β-果冻卷结构域的ProN-Tk-S359 A的整体结构与细菌前肽：枯草杆菌蛋白酶复合物的结构相似，不同之处在于它不含Ca 2+离子。为了分析Tk-SP的β-果冻卷结构域的作用，我们构建了一系列具有（Tk-S359 A/C）和不具有（Tk-S359 A/C Delta J）β-果冻卷结构域的Tk-SP活性位点突变体。Tk-S359 C和Tk-S359 C Delta J在凝胶测定中均表现出蛋白酶活性，表明β-果冻卷结构域不是折叠或活性所必需的。然而，在10 mM CaCl 2存在下通过远紫外CD光谱测定的Tk-S359 A Delta J的T-m值比Tk-S359 A低29.4 ℃。通过用10 mM乙二胺四乙酸处理，Tk-S359 A的T-m值降低了29.5 ℃，表明β-果冻卷结构域仅以Ca 2+结合形式有助于Tk-S359 A的稳定。Tk-SP在大小和氨基酸序列上高度类似于来自激烈火球菌、高温球菌和嗜热球菌的枯草杆菌蛋白酶样丝氨酸蛋白酶。我们认为，在C-末端连接一个β-果冻卷结构域是超嗜热菌蛋白适应高温环境的策略之一。(C)2010爱思唯尔有限公司保留所有权利。
Tk-SP is a hyperthermostable subtilisin-like serine protease from Thermococcus kodakaraensis and is autoprocessed from its precursor (Pro-Tk-SP) with N- and C-propeptides. The crystal structure of the active-site mutant of Pro-Tk-SP lacking C-propeptide, ProN-Tk-S359A, was determined at 2.0 angstrom resolution. ProN-Tk-S359A consists of the N-propeptide, subtilisin, and beta-jelly roll domains. Two Ca2+ ions bind to the beta-jelly roll domain. The overall structure of ProN-Tk-S359A without the beta-jelly roll domain is similar to that of the bacterial propeptide:subtilisin complex, except that it does not contain Ca2+ ions. To analyze the role of the beta-jelly roll domain of Tk-SP, we constructed a series of the active-site mutants of Tk-SP with (Tk-S359A/C) and without (Tk-S359A/C Delta J) beta-jelly roll domain. Both Tk-S359C and Tk-S359C Delta J exhibited protease activities in gel assay, indicating that the beta-jelly roll domain is not required for folding or activity. However, the T-m value of Tk-S359A Delta J determined by far-UV CD spectroscopy in the presence of 10-mM CaCl2 was lower than that of Tk-S359A by 29.4 degrees C. The T-m value of Tk-S359A was decreased by 29.5 degrees C by the treatment with 10 mM ethylenediaminetetraacetic acid, indicating that the beta-jelly roll domain contributes to the stabilization of Tk-S359A only in a Ca2+-bound form. Tk-SP highly resembles subtilisin-like serine proteases from Pyrococcus furiosus, Thermococcus gammatolerans, and Thermococcus onnurineus in size and amino acid sequence. We propose that attachment of a beta-jelly roll domain to the C-terminus is one of the strategies of the proteins from hyperthermophiles to adapt to high-temperature environment. (C) 2010 Elsevier Ltd. All rights reserved.